Pathogens and Disease, 79, 2021, ftaa069

doi: 10.1093/femspd/ftaa069
Advance Access Publication Date: 29 January 2021
Priority Paper

PRIORITY PAPER

How can we interpret SARS-CoV-2 antibody test

Downloaded from https://academic.oup.com/femspd/article/79/1/ftaa069/6123719 by guest on 18 March 2021

results?
Sofie Føns† and Karen A. Krogfelt*,‡
Roskilde University, Department of Science and Environment, Universitetsvej 1, Roskilde, Denmark
∗
Corresponding author: Department of Science and Environment, Roskilde University, Universitetsvej 1, 28A.1, DK-4000 Roskilde, Denmark. Tel/Fax: +45
46743269; E-mail: karenak@ruc.dk
One sentence summary: Antibody testing for SARS-CoV-2 is widespread despite poor test validation and limited knowledge on antibody responses,
which makes it crucial to understand both the potential, limitations and interpretation of serology.
Editor: Alfredo Garzino-Demo
†
Sofie Føns, http://orcid.org/0000-0003-0149-5909
‡
Karen A. Krogfelt, https://orcid.org/0000-0001-7536-3453

ABSTRACT
Since the outbreak of COVID-19, the world has raced to understand and accurately diagnose infection caused by
SARS-CoV-2. Today, hundreds of commercial antibody tests are on the market despite often lacking proper validation and
with unsatisfactory sensitivity and/or specificity. In addition, many questions related to the humoral response remain
unresolved, although research is carried out at an unprecedented speed. Despite the shortcomings, serological assays have
an important part to play in combating the pandemic by aiding in diagnosis and sero-epidemiological studies. However,
careful attention must be paid to the application of serology and the interpretation of serological data—especially in low
prevalence regions, both at an individual and at a population level. In this article, we argue that serological results are often
misinterpreted, and in the eagerness to be first, methodological rigor is often taking a backseat.

Keywords: coronavirus infections; serology; seroepidemiologic studies; antibodies; diagnosis; serologic tests

INTRODUCTION detection of past (or present) SARS-CoV-2 infection. As of 10th

of October 2020, the Foundation for Innovative New Diagnos-
Since the outbreak of COVID-19 in Wuhan in December 2019, the
tics lists 342 commercial immunoassays for detecting anti-
virus has, as of October 10th 2020, spread globally with 36 616 555
bodies (Foundation for Innovative New Diagnostics SARS-CoV-
confirmed cases and 1063 429 deaths worldwide (World Health
2 diagnostic pipeline 2020), but only 49 have currently been
Organization Coronavirus disease 2020). Following the release
granted an Emergency Use Authorization by the FDA (FDA 2020).
of viral genome sequences of SARS-CoV-2 in January (Zhang
The majority of these tests fall within two categories: either a
2020), molecular detection kits for real-time RT-PCR were soon
qualitative, rapid immunochromatographic assay (15–20 min),
developed and became the gold standard for diagnosing COVID-
or a slower semi-quantitative enzyme-linked immunoassay
19 by confirming the presence of SARS-CoV-2 RNA. The tests
(ELISA)/chemiluminescent immunoassay (CLIA) (a few hours).
have high specificities but varying sensitivities, mostly due to
Most commonly, they detect IgM, IgG or both antibodies, but
sampling difficulties, including choice of specimen, and tim-
some detect total antibody or IgA.
ing of peak viral load, which can lead to false-negative results.
Before long, however, companies, institutions and research lab-
oratories started flooding the market with serological kits for

Received: 11 August 2020; Accepted: 9 November 2020


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1
 2 Pathogens and Disease, 2021, Vol. 79, No. 1
Thorough validation is needed to facilitate the potential
of serology testing
Serology testing is a powerful way to monitor the progression of
the pandemic by seroprevalence studies and as a tool in diag-
nostics. For accurate diagnosis of COVID-19, serology can be a
great supplement to molecular detection. Serology is powerful
further into the course of the disease, when the virus has been
eliminated or exists in small numbers, as suggested in a number
of publications indicating antibody testing to surpass PCR sen-
sitivity 5–8 days after symptom onset (Guo et al. 2020; Yong et al.
2020; Zhao et al. 2020). However, in order to accurately use serol-
ogy for diagnostics or estimates of spread of infection in society,
extensive validation is needed. Many of the available tests are
of dubious quality, where especially the low specificity is of con-
cern.
Many manufacturers have not made their test validation
available and there are no standards to employ that make it
possible to compare the performance across tests and to make
the tests fully quantitative. Immunoassays vary on not only
which antibody they measure but also the antigen used, source
of the antigens, specimen type and the secondary antibody
conjugate, which influence the test performance (Haselmann
et al. 2020; Kontou et al. 2020; Schnurra et al. 2020). The need
for test harmonization is highlighted by the increasing num-
ber of studies published that compare the head-to-head per-
formance of immunoassays (GeurtsvanKessel et al. 2020; Har-
ritshoej et al. 2020; Jääskeläinen et al. 2020; Lassaunière et al.
2020; Schnurra et al. 2020; Whitman et al. 2020), often show-
ing some discrepancy. Those studies have used pre-pandemic
sera, some of which were samples from patients with respira-
tory virus infections, as it is essential to be able to discriminate
between the e.g. ‘common cold’ coronaviruses and SARS-CoV-2
to avoid false positives.
An additional concern is the potential batch-to-batch varia-
tion between tests, which leads to the need for repeated valida-
tion for each batch used. In Denmark, the study of seropreva-
lence among blood donors had to be halted, as a new batch of
the IgM/IgG Antibody to SARS-CoV-2 lateral flow test from Livzon
Diagnostics showed remarkably lower sensitivity than previous
batches (Leverance af antistoftest 2020).
What do sensitivity and specificity tell us? Interpreting
an individual test result
The high number of antibody tests on the market each has a dif-
ferent sensitivity and specificity. A highly sensitive test should
capture all true positive results, whereas a highly specific test
should rule out all true negative results. In reality, none of the
tests are both 100% sensitive and specific, hence the importance
of validating the test before use to know the test characteris-
tics. The test results from a population-based serology survey
can then be adjusted for the imperfect test quality. One concern
related to validation is what kind of samples were used as pos-
itive controls. Do they reflect the population being surveyed? If
not, we might underestimate the seroprevalence. The positive
control samples are from PCR-confirmed COVID-19 patients, but
they might not represent the full clinical spectrum or the differ-
ent age groups. It is still not known whether children in gen-
eral have a different pattern of antibody generation compared
to adults with COVID-19, and in addition, the severity of disease
affects the antibody response, thus samples of asymptomatic
ought to be included in the validation.
A general trend seems like that the rapid tests tend to have
lower sensitivity than the semi-quantitative tests (Kontou et al.
2020), thus underestimating the true rate of seroconversion in
those tested. An advantage of the rapid tests is their speed and
ease of use that does not require a laboratory. However, they do
depend on the operator to interpret whether they are positive or
not, typically by the visualization of a red line, which can result
in borderline cases.
Despite the wide spread of SARS-CoV-2, most areas around
the world still have an overall low seroprevalence, which poten-
tiates the problem of false positives when deploying antibody
tests. Even in a hard-hit country like Spain, findings from per-
haps the most extensive population-based sero-epidemiological
study to this day, suggests that only 5% of the population had
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antibodies against SARS-CoV-2 (Pollán et al. 2020).
But how does the seroprevalence impact the interpretation of
an individual test result? Let us take an example. In Denmark,
a study among 20 640 blood donors showed an adjusted sero-
prevalence of 1.9% (Erikstrup et al. 2020). The sensitivity of the
test was estimated to be 82.6% and specificity 99.5%. These fig-
ures result in a negative predictive value of 99.7% and a posi-
tive predictive value of 76.2%. Given a negative result as a blood
donor, the probability that the result is right is almost 100%. Is
the result of your test positive, the probability that the result is
correct is only about three-quarters. Are you, as an individual,
much better off knowing your antibody status than before? Prob-
ably not. If you live in an area with low seroprevalence and you
feel healthy, the chances of you having had COVID-19 was small
anyway, whereas a positive result has an almost 25% chance
of being false. On top of that, we still do not know whether a
positive antibody test is associated with protection from future
COVID-19 infection and we also do not know for how long the
antibodies last, so in fact you should not act any differently than
if you had a negative result.
The issue of a low positive predictive value is potentiated
the lower the seroprevalence, and thus underscores the chal-
lenges of accurately assessing one’s antibody status in areas so
far spared from big outbreaks of SARS-CoV-2–despite using a
test with a seemingly high specificity. An alternative approach
to increase the positive predictive value is to focus testing on
individuals with an elevated likelihood of previous exposure to
SARS-CoV-2 e.g. a history of COVID-19-like illness, or employ a
second test with different design characteristics (e.g. antibody
format or antigen) if the first test was positive [(CDC Information
for Laboratories about Coronavirus (COVID-19) 2020; Hicks et al.
2020)]. As previously mentioned, further into the course of the
disease, serology testing is likely more sensitive than molecu-
lar methods, and integration of different testing methods could
help ensure correct and timely diagnosis of COVID-19. In cer-
tain areas without access to advanced laboratories, rapid anti-
gen testing, although typically less sensitive than RT-PCR, could
also be a relevant alternative e.g. for screening (CDC Information
for Laboratories about Coronavirus (COVID-19) 2020).
Kinetics of SARS-CoV-2 antibody response
By using an ELISA or other semi-quantitative tests, testing
COVID-19 cases can potentially reveal something about the
kinetics of the antibody response. Despite often being consid-
ered a marker of acute infection, IgM does not consistently
appear before its IgG counterpart, which hinders its use as a
marker of acute or recent infection. A similar trend for IgM was
found among studies of SARS-CoV (Meyer, Drosten and Müller

2014), but it could partly be due to differences in testing sensi-
tivities. The median seroconversion reported in several studies
falls between 9 and 14 days post symptom onset (Grzelak et al.
2020; Long et al. 2020; Lou et al. 2020; Qu et al. 2020; Zhao et al.
2020), which emphasizes the importance of timing when testing
for antibodies. One important point to make is the variability in
the antibody response with some patients seroconverting within
a few days post symptom onset, and others taking weeks to do
so, thus testing too early will miss some cases. Testing for total
antibodies appears to be more sensitive and thus detectable a
little earlier than IgM or IgG alone (Harritshoej et al. 2020; Las-
saunière et al. 2020; Lou et al. 2020; Zhao et al. 2020). IgA specific
tests are rare, but some studies report a potential use of IgA as
an early diagnostic marker (Dahlke et al. 2020; Ma et al. 2020).
Two large studies found that IgG antibodies persisted for at
least three to four months after symptom onset (Gudbjartsson
et al. 2020; Iyer et al. 2020), although other studies have observed
a gradual decline within the first couple of months (Long et al.
2020; Perreault et al. 2020; Wang et al. 2020). Quantitative mea-
surement of antibody titers also makes it possible to look for cor-
relation to severity status of COVID-19 patients (PCR confirmed),
with a range of studies finding higher titers among severe cases
(Liu et al. 2020; Long et al. 2020; Qu et al. 2020; Salazar et al. 2020),
however, the causality is still unclear. Is it because of higher viral
load? Is it because the virus has successfully invaded and colo-
nized the host? Or is the immune response detrimental?
One overall problem though is the lack of proper longitudinal
studies, although with time passing since the outbreak of the
pandemic more studies are surfacing (Iyer et al. 2020; Perreault
et al. 2020; Wang et al. 2020). Most serology studies to this date
are retrospective or cross-sectional, and those of longitudinal
character often have few patients and/or few sequential sam-
ples, which limit their use for accurately answering outstanding
issues regarding antibody kinetics.
Comparisons of molecular testing followed by antibody test-
ing show that most individuals with symptoms seroconvert and
that PCR testing can be positive up to a month after symptom
recovery (Wajnberg et al. 2020). However, neither the clinical fea-
tures nor the immune responses of asymptomatic cases have
been well described yet. So far, most studies have focused on
hospitalized, PCR confirmed COVID-19 patients and their anti-
body response. But some people may fail to mount a detectable
antibody response altogether. A small study found that asymp-
tomatic cases may have a weaker immune response to the virus
and that the antibodies may diminish sooner than for symp-
tomatic cases with a reduction in neutralizing antibodies after
eight weeks (Long et al. 2020).
It is still unclear what antigen(s) are preferred in
antibody assays
An important aspect to discuss is the impact of antigens in sero-
logical testing. By far the most common antigens to use are the
structural proteins nucleocapsid (N) and spike (S) protein, which
are also the most immunogenic. The N protein is the most abun-
dant protein; it is small and can readily be expressed in e.g. E. coli.
On the other hand, the trimeric spike protein extrude from the
surface and the S1 subunit is used for receptor binding through
the individually folded receptor binding domain (RBD), which is
likely a primary target for neutralizing antibodies (Wrapp et al.
2020). The S protein is heavily glycosylated and is therefore typ-
ically expressed in mammalian cells. Many antibody kits make
use of only one antigen, which opens for the possibility that
Føns and Krogfelt 3
some individuals might not have a strong antibody response
towards that particular antigen. Other kits only use part of the
S protein, e.g. the RBD, again possibly introducing a selection
bias. The use of recombinant antigens leads to less biosafety
needed, it is more standardized and perhaps cross-reactivity can
be avoided if only specific epitopes on the viral proteins are used.
A few research groups have developed peptide or protein
microarrays, which could help establish the level of cross-
reactivity between antigens and which antigens elicit the
strongest response (Jiang et al. 2020). However, peptide microar-
rays come with a risk of false negatives if the antibodies only rec-
ognize conformational epitopes instead of linear. The N protein,
like the S2 subunit of the spike, is more conserved across coro-
naviruses, which may increase the risk of cross-reactivity. One
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study found that seroconversion occurred in average two days
earlier for assays detecting total Ig or IgG anti-N than for IgG
anti-S (Van Elslande et al. 2020), however another study found
more patients had earlier seropositivity for anti-RBD (To et al.
2020). Studies comparing the use of different antigens point in
different directions with some concluding that N is preferred,
others that S1 subunit or RBD is the more specific and sensi-
tive choice (GeurtsvanKessel et al. 2020; Jiang et al. 2020; Liu et al.
2020; Ma et al. 2020; Schnurra et al. 2020; To et al. 2020).
Correlates of protection—are we any wiser 10 months
into the pandemic?
Often when the detection of an antibody response towards
SARS-CoV-2 is discussed, it is assumed that reactivity correlates
with neutralization, and that neutralization equals immunity
(or confers some level of protection), which like WHO warned
in April, is too early to say. There are different assays com-
monly employed when testing for neutralization. Plaque reduc-
tion neutralization test (PRNT) is considered the gold standard;
however, like the cytopathic effect-based microneutralization
(MN) assay, it makes use of cultivated live virus that requires a
biosafety lab level 3 (BSL-3). Instead, many researchers make use
of pseudotyped neutralization assays, which can be handled in
a BSL-2 lab. Pseudotyped virus neutralization assays have been
used for many types of viruses, however, few SARS-CoV-2 stud-
ies have examined its correlation to other neutralization assays
like PRNT or MN (Grzelak et al. 2020). A series of studies have
reported a correlation between detecting antibodies or antibody
titers to neutralizing ability (GeurtsvanKessel et al. 2020; Grzelak
et al. 2020; Jääskeläinen et al. 2020; Salazar et al. 2020; To et al.
2020; Wu et al. 2020), but binding is not always predictive of neu-
tralization (Criscuolo et al. 2020; Manenti et al. 2020).
Despite the uncertainty of the role of neutralizing antibod-
ies and the waning of protection, we can probably draw on our
knowledge from other viral infections. We can likely expect that
we are either immune against reinfection for months or per-
haps even a couple of years, or that having encountered the
virus before at least will help clear the virus faster the next
time around with possibly fewer symptoms. From the SARS epi-
demic back in 2003 we know that high antibody levels are main-
tained for at least 16 months before declining significantly (Liu
et al. 2006), but one study found that some patients still had
detectable neutralizing antibodies 17 years later (Anderson et al.
2020). The humoral response is not the only level of protec-
tion, so studies on the cellular immunity are also warranted.
In a study by Braun et al., they found that 83% of COVID-19
patients as well as 34% of healthy donors had SARS-CoV-2 spike
protein-reactive CD4+ T-cells, albeit at lower frequencies among
 4 Pathogens and Disease, 2021, Vol. 79, No. 1
the healthy donors (Braun et al. 2020). It was speculated that this
might correlate to some protection if you have had a common
cold from coronaviruses. Other studies have similarly observed
T-cell reactivity against SARS-CoV-2 in unexposed people, but
the source and clinical relevance remain unknown (Sette and
Crotty 2020). Single cell transcriptomic analysis has helped shed
light on the remarkable heterogeneity in the SARS-CoV-2 reac-
tive CD4 + T cell response among patients with subsets of T-cells
correlating to disease severity and antibody levels (Meckiff et al.
2020).
Recently, confirmed cases of reinfection have been reported
in various countries (Gupta et al. 2020; Tillett et al. 2020; To et al.
2020), however, this is not necessarily a big concern nor unex-
pected. Waning antibody levels, a poorly developed immune
response to SARS-CoV-2 from the first infection or genetic
changes in the viral surface antigens could be the explanation.
These reinfection cases may be outliers, or reinfection may be
more common for other infections as well than we know due
to less scrutiny compared to SARS-CoV-2. It is important to note
though that a decline in antibody levels after a few months since
symptom onset is normal and does not rule out the longevity of
protection, as it is also conferred by memory cells.
Literature on COVID-19 is exploding, but warrants a
word of caution
On a final note, it is challenging to stay aware of all the literature
relating to SARS-CoV-2 serology. The number of publications is
exploding and preprints are being released at an unprecedented
speed. As of October 11th 2020, the preprint servers medRxiv and
bioRxiv contain 9456 articles related to COVID-19/SARS-CoV-2
(medRxiv COVID-19 2020). On one hand, such a unified response
by the scientific community is remarkable, on the other hand, it
is becoming increasingly difficult for proper science to stand out
and some studies and manuscripts are likely rushed.
Additionally, in the eagerness of making preliminary results
readily accessible, the outcome of many of the earliest sero-
prevalence studies were reported in the press before a scientific
(albeit not necessarily peer-reviewed) article was released. That
is problematic since such results might influence public policy
and public opinion before the scientific community has had the
chance to scrutinize the results and methods. Often these stud-
ies were based on convenience sampling with a selected group
and/or had a low participation rate, and thus they were not rep-
resentative for the general population. However, the results from
large serological studies like the Spanish ENE-COVID are now
appearing (Pollán et al. 2020).
CONCLUSION
We have a lot left to learn about the antibody response to SARS-
CoV-2 infection and how this knowledge can guide us in our
efforts to combat the pandemic. When using serological assays,
careful consideration must be placed on the testing strategy
with focus on maximizing specificity and consequently positive
predictive value, since the overall prevalence of antibodies in
most populations is still low.
ACKNOWLEDGEMENT
We thank Lukas Ocias for critical reading of the manuscript and
Lone Simonsen (LS) as well as the PandemiX research team for
fruitful discussions.
FUNDING
This work was supported by the Lundbeck Foundation [R349-
2020-703 to KAK] and the Carlsberg Foundation [CF20-0046 to
LS].
Conflicts of interest. None declared.
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